Due to their tunable physicochemical properties, peptides are capable of folding into compact structural motifs. These shapes can form nanosized architectures in the shape of tubes, micelles, monolayers and bilayers.
Lysine and cysteine peptides are used in DPRA as a model to evaluate the molecular initiating event of a chemical substance, framed by OECD Guideline no 442. The peptides can be immobilized in electrochemical biosensors to detect various quantities such as current (amperometric and voltammetric) or potential.
The DPRA assay is an in chemico test method that aims to detect chemicals able to react with and covalently attach to the amino acid residues of human skin proteins. It is based on the assumption that most chemical allergens are small molecules with electrophilic properties that can react with electron-rich groups of the nucleophilic amino acids in the proteins, resulting in the formation of haptens. The DPRA assay simulates this process in a non-cellular system, allowing the detection of possible sensitizers by quantifying their reactivity towards synthetic peptides (Chipinda et al, 2011).
The DPRA method is currently under evaluation for its ability to predict whether chemicals are likely to be skin sensitizers and has been incorporated into an OECD test guideline (TG442C). However, some concerns about its predictive performance remain, especially when tested with mixtures. A recent study investigated nine compounds in a surrogate mixture containing two known skin sensitizers and one known non-sensitizer. The DPRA resulted in correct classification for eight of the nine compounds, even though they were tested at significantly lower concentrations than their EC3 values (0.4 mM) and did not undergo the recommended molar excess conditions of 100 mM. Only the extremely potent skin sensitizer oxazolone was not correctly classified, but this could have been due to the low number of peptide depletion measurements that were made.
An alternative to the DPRA assay is the kinetic direct peptide reactivity assay (kDPRA), which also identifies chemicals that react with and deplete the peptides. The assay measures the kinetics of the interaction between the peptide and a test chemical by measuring the percentage depletion over time, producing a data matrix. The kDPRA parameter logkmax is calculated from this data matrix and assigns chemicals to the 1A potency class if logkmax reaches the threshold value of -2.
The DPRA and kDPRA are currently being combined with predictions from in vitro cell-based models to predict the overall sensitisation potential of chemicals. These predictions are then used to inform decisions about the need for further testing and for the selection of suitable non-animal test methods.
Molecular Initiating Event
The molecular initiating event (MIE) is the initial point of chemical-biological interaction. The MIE may be a direct binding of a chemical to a protein or it may cause an indirect pathway, such as induction of gene expression. The MIE can be predicted based on the chemistry of the chemicals and their properties, allowing for in silico prediction models. This MIE-based approach is called adverse outcome pathway (AOP) modeling and is an alternative to animal testing.
An AOP is a mechanistic model of biological processes connecting chemical exposures to harmful effects. It consists of a sequence of events connecting molecular interactions at different levels of biological organization, including cellular and organ responses. AOPs are a new framework for chemical risk assessment that complements, rather than replaces, existing animal test data.
AOPs can provide guidance for the development of new test methods by linking specific chemical-biological interactions with biological endpoints relevant to human health and environmental safety. They can also help to avoid unnecessary animal tests and support a sustainable business model. AOPs are a key component of the shift away from traditional animal-based testing towards new approaches.
For example, the AOP for skin sensitization focuses on the molecular initiating event of covalent binding of a chemical to proteins in the skin. It leads to downstream key events at the molecular and cellular level that can lead to an adverse outcome, such as erythema or inflammation. The AOP framework allows for the prediction of these key events based on a chemical’s chemistry, which is measured in DPRA tests and other in vitro tests.
This AOP model can be used to determine the optimal concentration of a chemical required for induction of an MIE in human cells in a lab experiment. The model can also be used to design more targeted chemical screens for the detection of potential MIEs in humans.
In this study, two intersecting AOPs (AOP 347 and AOP 347), involving the MIEs of peroxisome proliferator-activated receptor-g inactivation and Toll-like receptor 4 activation, were modeled using a number of MIE modeling techniques including molecular dynamics, pharmacophore models, and quantitative structure–activity relationship models. The resulting prediction models showed high accuracies and were validated by in vitro experiments.
Allergies are a response by the immune system to foreign proteins, usually proteins found in food, dust mites, pollen, some chemicals and fungi such as yeast. Normally, the first exposure to these substances does not cause a reaction. But in people who are allergic to a particular protein, the first exposure triggers an immune response that results in the body producing large amounts of antibodies to the allergen. These antibodies bind to cells that live in the skin, the respiratory tract (airways) and the hollow organs that connect from the mouth to the anus (gastrointestinal or GI tract). The cells then release chemicals such as histamine. These chemicals irritate tissues, leading to inflammation and itching.
Most allergens are proteins or glycoproteins, but certain rare instances involve pure carbohydrates or low-molecular-weight chemical metabolites such as salts and pigments. Allergenic proteins share significant amino acid sequence homology. Proteins from the same species produce similar allergenic proteins that are grouped together under a single allergen name. The Allergen Nomenclature Subcommittee of the World Health Organization and International Union of Immunological Societies maintains an extensive database of known allergens.
Upon the first exposure to an allergen, the immune system produces antibodies that bind to cells called mast cells. These cells are located in the skin, the lining of the respiratory tract and the gastrointestinal tract. The mast cells then release chemicals such as histamine, leading to an allergic reaction in the tissues and mucus membranes of the affected organs.
Many allergens are proteins, such as a protein from cockroaches that causes a sensitivity to pollen, or a protein from milk called b-lactoglobulin. Other proteins that are the cause of allergies include a heparin-binding protein from a fungus, a family of proteins called calycins, first identified as being responsible for hay fever, and ligand-binding proteins such as der p 1 from grasses, which activate IgE antibody responses.
Other types of allergens are chemicals and dietary additives. For example, some people are allergic to lecithin, an emulsifier that may contain eggs or soybeans. Also, people can develop an allergic reaction to carrageenan, a thickening agent made from red seaweed.
Peptides are synthetic molecules that consist of 20 amino acids or less. They are assembled by peptide linkers which connect amino acid residues of the desired sequence. The sequence of the peptide is determined by selecting one or more reagents to protect and activate the peptide bonding site for each amino acid residue in the peptide sequence (Borgia and Fields 2000). Peptide synthesis has improved and has become more efficient with faster coupling reagents and lower temperature reaction conditions. These improvements have also allowed a higher degree of control over peptide production to ensure that each peptide is constructed in accordance with its chemical structure.
The biological activity of peptides can be attributed to the fact that they have several key attributes, such as their cationic nature and amphipathic structure which enables them to have both positively charged and hydrophobic faces. These properties allow peptides to interact with the outer membrane of cells, causing damage and subsequent cell death. They have also been shown to act directly on the viral envelope. This type of action is mediated through binding the peptide to the virus or to specific receptors on the host cell.
Cationic peptides, such as iui> 1/4-MSH, have been shown to have antimicrobial activity by targeting the surface of the bacteria or microorganism (Jenssen and others 2006). The mechanism involves electrostatic interaction between the peptide and the negatively charged outer membrane components, such as lipopolysaccharides in Gram-negative bacteria and lipoteichoic acid in Gram-positive bacteria, resulting in the depletion of divalent ions on the cell membrane, allowing entry of the peptide into the cytoplasmic membrane.
A number of other studies have demonstrated that peptides can have antiviral and antifungal activities. Antifungal peptides have been shown to be effective by targeting and penetrating the fungal cell membrane, which results in a loss of membrane integrity. In particular, peptides that have been derived from the sequence of cathelicidins have been shown to be effective at targeting and damaging the plasmatic membrane of the fungus Trichosporon beigelii.
Biological activity of peptides can be evaluated by using a variety of techniques, including immunochemistry and scanning electron microscopy (SEM). These methods have been used to evaluate morphological changes in polymeric films that have been incorporated with peptides. These changes, in turn, are a direct reflection of the peptide’s bioactivity. direct peptides